本文關(guān)鍵詞：關(guān)于炎癥因子IL-6和血管緊張素II調(diào)節(jié)內(nèi)皮酯酶表達(dá)的信號(hào)傳導(dǎo)通路的研究　出處：《山東大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 關(guān)于 炎癥 因子 IL-6 血管 緊張 調(diào)節(jié) 內(nèi)皮 酯酶 表達(dá) 信號(hào) 傳導(dǎo) 通路 研究

【摘要】：背景和目的 目前臨床上在脂質(zhì)代謝紊亂方面的治療還主要集中在降低血漿低密度脂蛋白膽固醇(low-density lipoprotein cholesterol, LDL-C)水平方面。降低LDL-C水平減少罹患心血管疾病風(fēng)險(xiǎn)的觀點(diǎn)已經(jīng)得到廣泛認(rèn)同。然而臨床結(jié)果顯示經(jīng)過(guò)他汀類藥物標(biāo)準(zhǔn)治療后即使LDL-C達(dá)標(biāo),仍有10.9%的心血管剩留風(fēng)險(xiǎn)。治療新靶點(diǎn)研究(TNT)顯示,經(jīng)過(guò)他汀類藥物強(qiáng)化治療后即使LDL-C水平降至1.99mmol/L,明顯低于達(dá)標(biāo)水平,仍有8.7%的絕對(duì)冠狀動(dòng)脈事件剩留風(fēng)險(xiǎn),提示即使經(jīng)過(guò)他汀類藥物強(qiáng)化治療,冠狀動(dòng)脈事件剩留風(fēng)險(xiǎn)仍較高。血脂異常相關(guān)的心血管剩留風(fēng)險(xiǎn)與多種因素有關(guān),以低高密度脂蛋白(high-density lipoprotein cholesterol, HDL-C)為特征的血脂異常是經(jīng)過(guò)他汀類藥物治療降低LDL-C水平后最常見的其中一種。如何降低冠狀動(dòng)脈事件剩留風(fēng)險(xiǎn),減少心血管事件發(fā)生率,也是目前研究的焦點(diǎn)之一。HDL-C水平與心血管發(fā)生率呈負(fù)相關(guān),約有1/3的血脂異�；颊咄ㄟ^(guò)提高HDL-C水平而受益,有研究表明血漿HDL-C每升高0.03mmol/L(1.0mg/dl)就可以使罹患心血管疾病的風(fēng)險(xiǎn)下降2%-4%。另外LDL-C/HDL-C比值也是一個(gè)與心血管事件密切相關(guān)而獨(dú)立于LDL-C和HDL-C的重要指標(biāo)。因此治療動(dòng)脈粥樣硬化除了在一定范圍內(nèi)降低LDL-C水平之外如何有效的提高HDL-C水平就成為了重要的研究方向。 內(nèi)皮脂酶(endothelial lipase, EL)是近年新發(fā)現(xiàn)的甘油三酯脂肪酶家族新成員,直接由血管內(nèi)皮細(xì)胞分泌,在局部發(fā)揮作用。主要具有磷脂酶活性,是代謝HDL-C的關(guān)鍵酶,如何能夠通過(guò)減低或抑制EL活性,從而減少HDL-C的降解,是治療動(dòng)脈粥樣硬化的新發(fā)展思路。因此研究EL的轉(zhuǎn)錄表達(dá)調(diào)節(jié),如何有效的降低EL就顯得極為迫切。 血管緊張素Ⅱ(Angiotensin Ⅱ, Ang Ⅱ)和炎癥因子白細(xì)胞介素-6(interleukin-6,IL-6)是兩種非常重要的致動(dòng)脈粥樣硬化危險(xiǎn)因素。Ang Ⅱ能通過(guò)增加巨噬細(xì)胞清道夫受體CD36,促進(jìn)巨噬細(xì)胞攝取氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL),加速泡沫細(xì)胞形成；能通過(guò)誘導(dǎo)炎癥反應(yīng)及細(xì)胞凋亡、產(chǎn)生氧自由基和影響纖溶功能等多方面參與動(dòng)脈粥樣硬化的病理過(guò)程;可以增加斑塊不穩(wěn)定因子[7]EMMPRIN和MMPs[8、9]的表達(dá),加重動(dòng)脈粥樣硬化病變。IL-6也是一種重要的致動(dòng)脈粥樣硬化炎癥因子,它構(gòu)成了許多急慢性疾病的病理學(xué)基礎(chǔ),能夠損傷內(nèi)皮細(xì)胞造成內(nèi)皮細(xì)胞功能障礙,增加單核-內(nèi)皮細(xì)胞的粘附,還可以促進(jìn)巨噬細(xì)胞對(duì)脂質(zhì)的攝取。核因子NF-κB是種重要的核轉(zhuǎn)錄調(diào)節(jié)因子,激活后可啟動(dòng)包括細(xì)胞因子、化學(xué)因子等多種效應(yīng)基因的表達(dá)。絲裂原活化蛋白激酶MAPKs級(jí)聯(lián)反應(yīng)是細(xì)胞內(nèi)重要的信號(hào)傳導(dǎo)系統(tǒng)之一,參與多種胞內(nèi)信息傳遞過(guò)程,能對(duì)廣泛的細(xì)胞外刺激發(fā)生反應(yīng),研究表明,NF-κB和p38MAPK在動(dòng)脈粥樣硬化中表達(dá)增加，可能是各種危險(xiǎn)因子誘發(fā)動(dòng)脈粥樣硬化的機(jī)制之一,NF-κB和p38MAPK信號(hào)傳導(dǎo)途徑可能在EL的轉(zhuǎn)錄表達(dá)過(guò)程中發(fā)揮作用。本課題通過(guò)體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞,以IL-6和Ang Ⅱ刺激內(nèi)皮細(xì)胞,觀察IL-6、Ang Ⅱ、NF-κB p65阻斷劑PDTC (Pyrrolidinedithioearbamic acid)和SB203580(p38MAPK抑制劑)對(duì)EL表達(dá)的影響,驗(yàn)證IL-6和AngⅡ是否通過(guò)NF-κB和MAPK信號(hào)傳導(dǎo)途徑調(diào)節(jié)EL的表達(dá)。 研究方法 1.提取新生兒臍靜脈內(nèi)皮細(xì)胞進(jìn)行原代培養(yǎng),經(jīng)鑒定為內(nèi)皮細(xì)胞后,貼壁法傳代培養(yǎng)。第四代細(xì)胞用于實(shí)驗(yàn)。 2.用于實(shí)驗(yàn)的人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells.HUVKCs)共分六組①AngⅡ刺激組：在培養(yǎng)液中加入AngⅡ,使之終濃度為10umol/L;②四氫化吡咯烷二硫代氨基甲酸酯(PDTC)+AngⅡ刺激組,在培養(yǎng)液中加入PDTC使之終濃度為10mmol/L,預(yù)處理1h后加入AngⅡ (10umol/L)刺激;③SB203580+AngⅡ (10umol/L)刺激組,在培養(yǎng)液中加入SB203580使之終濃度為10imol/L,預(yù)處理1h后加入AngⅡ(10umol/L)刺激；④IL-6刺激組：在培養(yǎng)液中加入rhIL-6,使之終濃度為10ng/mL,預(yù)處理1h后加入AngⅡ(10umol/L)刺激;⑤四氫化吡咯烷二硫代氨基甲酸酯(PDTC)+IL-6刺激組,在培養(yǎng)液中加入PDTC,使之終濃度為10mmol/L,預(yù)處理1h后加入rhIL-6(10ng/ml)刺激;⑥SB203580(10umol/L)+IL-6刺激組,在培養(yǎng)液中加入SB203580使之終濃度為10umol/L,預(yù)處理1h后加入IL-6(10ng/mL)刺激。各組分別孵育0、2、4、8、12、24h后終止實(shí)驗(yàn),收集細(xì)胞。 3.將收集的細(xì)胞提取蛋白,使用western blot法檢測(cè)EL的表達(dá)。 4.以上數(shù)據(jù)使用統(tǒng)計(jì)學(xué)進(jìn)行分析。 結(jié)果 1. AngⅡ可以上調(diào)HUVECs EL的表達(dá)。HUVECs經(jīng)AngⅡ刺激后,在4,8,12h時(shí)其EL的表達(dá)量分別為0.362±0.041,0.738±0.034和0.387±0.051,均較0h(0.254±0.027)時(shí)明顯增加,均p0.05。 2. PDTC可以抑制由AngⅡ引起的EL表達(dá)的增高。HUVECs經(jīng)PDTC預(yù)處理1h后再加入AngⅡ刺激,其EL表達(dá)量在作用4,8,12h時(shí)分別為0.287±0.037,0.433±0.041,0.303±0.025,與AngⅡ刺激組在相應(yīng)時(shí)間段內(nèi)EL的表達(dá)量相比明顯降低,均p0.05。 3.SB203580可以抑制由AngⅡ引起的EL表達(dá)的增高。HUVECs經(jīng)SB203580預(yù)處理1h后再加入AngⅡ刺激,其EL表達(dá)量在作用4,8,12h時(shí)分別為0.258±0.027,0.372±0.038,0.296±0.034,與AngⅡ刺激組在相應(yīng)時(shí)間段內(nèi)EL的表達(dá)量相比明顯降低,均p0.05。 4.IL-6可以上調(diào)HUVECs EL的表達(dá)。HUVECs經(jīng)IL-6刺激后,在4,8,12h時(shí)其EL的表達(dá)量分別為0.293±0.062,0.633±0.052,0.462±0.065,均較Oh(0.254±0.027)時(shí)明顯增加,均p0.05。 5. PDTC可以抑制由IL-6引起的EL表達(dá)的增高。HUVECs經(jīng)PDTC預(yù)處理1h后再加入AngⅡ刺激,其EL表達(dá)量在作用4,8,12h時(shí)分別為0.208±0.056,0.582±0.036,0.428±0.066,與IL-6刺激組在相應(yīng)時(shí)間段內(nèi)的EL表達(dá)量相比明顯降低,均p0.05。 6.SB203580可以抑制由IL-6引起的EL表達(dá)的增高。HUVECs經(jīng)SB203580預(yù)處理1h后再加入AngⅡ刺激,其EL表達(dá)量在作用4,8,12,24h時(shí)分別為0.238±0.023,0.508±0.058,0.423±0.052,0.199±0.042,與IL-6刺激組在相應(yīng)時(shí)間段內(nèi)的EL表達(dá)量相比明顯降低,均p0.05。 結(jié)論 1. AngⅡ可能通過(guò)NF-κB和p38MAPK信號(hào)傳導(dǎo)通路促進(jìn)人臍靜脈內(nèi)皮細(xì)胞中內(nèi)皮脂肪酶的表達(dá)。 2.IL-6可能通過(guò)NF-κB和p38MAPK信號(hào)傳導(dǎo)通路促進(jìn)人臍靜脈內(nèi)皮細(xì)胞中內(nèi)皮脂肪酶的表達(dá)。
[Abstract]:Background and purpose
At present the clinical treatment of lipid metabolic disorder in the area is mainly concentrated in the lower plasma low density lipoprotein cholesterol (low-density lipoprotein cholesterol, LDL-C) level. Reduce the level of LDL-C has been widely recognized by the risk of cardiovascular disease. However, the idea of reducing the clinical results after statin drugs after treatment even if the LDL-C standard standard, there are still 10.9% the residual cardiovascular risk. A new target for the treatment of (TNT) showed that after intensive statin therapy. Even after the level of LDL-C to 1.99mmol/L was significantly lower than the standard level, there are still 8.7% of the absolute residual risk of coronary events, suggesting that even after intensive statin therapy, coronary events residual risk is still high. Dyslipidemia related residual cardiovascular risk associated with many factors, with low high density lipoprotein (high-density lipoprotein choleste Rol, HDL-C) is characterized by abnormal blood lipid after statin therapy reduces the level of LDL-C after the most common one. How to reduce the residual risk of coronary events, reduce the incidence of cardiovascular events is currently one of the research focus in the.HDL-C level and cardiovascular incidence were negatively correlated, about 1/3 of patients with dyslipidemia the level of HDL-C and benefit, studies have shown that plasma HDL-C increased 0.03mmol/L (1.0mg/dl) can be an important indicator of the risk of cardiovascular disease by 2%-4%. and ratio of LDL-C/HDL-C is a closely related with cardiovascular events independently of LDL-C and HDL-C. So the treatment of atherosclerosis in addition to reduce the level of LDL-C in a certain range from how to effectively improve the level of HDL-C has become an important research direction.
Endothelial lipase (endothelial lipase EL) is a new member of the family recently discovered triglyceride lipase, secreted by endothelial cells, play a role in the local. The main with phospholipase activity, is a key enzyme in the metabolism of HDL-C, how to reduce or inhibit the activity of EL, thereby reducing the degradation of HDL-C, is a new development way of treating atherosclerosis the expression of EL. So the study of transcriptional regulation, how to effectively reduce the EL is extremely urgent.
Angiotensin II (Angiotensin II, Ang II) and inflammatory cytokines interleukin -6 (interleukin-6, IL-6) are two important risk factors of atherosclerosis.Ang II by increasing macrophage scavenger receptor CD36, promote macrophage uptake of oxidized low density lipoprotein (oxidized low density lipoprotein, ox-LDL), the formation of acceleration foam cells; can induce inflammatory reaction and apoptosis, the pathological process of oxygen free radicals and influence of fibrinolytic function and other aspects involved in atherosclerosis; plaque instability could increase the expression of factor [7]EMMPRIN and MMPs[8,9], increased atherosclerosis in.IL-6 is also an important factor of atherosclerosis, which constitutes the pathology of many acute and chronic diseases the science foundation, can damage endothelial cells caused by endothelial dysfunction, increased monocyte and endothelial cells The adhesion, can also promote the uptake of lipid in macrophage. Nuclear factor kappa B NF- is an important regulator of nuclear transcription factor, activator can activate the expression of a variety of effects including cytokines, chemical factors genes. MAPKs mitogen activated protein kinase cascade is one of the important intracellular signal transduction systems, participate in a variety of intracellular information the transfer process, can on a wide range of extracellular stimuli, studies show that NF- kappa B and increase the expression of p38MAPK in atherosclerosis, may be one of the mechanisms of various risk factors for atherosclerosis, NF- kappa B and p38MAPK signaling pathway may play a role in the process of expression in the transcription of EL. The in vitro cultured human umbilical the venous endothelial cells with IL-6 and Ang II stimulated endothelial cells, observed IL-6, Ang II, NF- kappa B p65 inhibitor PDTC (Pyrrolidinedithioearbamic acid) and SB203580 (p38M The effect of APK inhibitor on the expression of EL verifies whether IL-6 and Ang II regulate the expression of EL by means of NF- - kappa B and MAPK signal transduction pathway.
research method
1. the umbilical vein endothelial cells of the newborn were extracted and cultured. After being identified as endothelial cells, the endothelial cells were cultured and cultured. The fourth generation cells were used for the experiment.
2. experiments on human umbilical vein endothelial cells (human umbilical vein endothelial cells.HUVKCs) were divided into six groups: group Ang II stimulation was added into the culture fluid of Ang II, the final concentration of 10umol/L; the four hydrogenated pyrrolidine two thiocarbamate (PDTC) stimulated by +Ang in the group, added to the culture medium PDTC to the final concentration of 10mmol/L, pretreatment of 1h after adding Ang II (10umol/L) stimulation; the SB203580+Ang II (10umol/L) stimulation group, in the culture medium added SB203580 to a final concentration of 10imol/L, pretreatment of 1h after adding Ang II (10umol/L) and IL-6 stimulation; stimulation group: rhIL-6 in cultures in the end, the concentration of 10ng/mL, pretreatment of 1h after adding Ang II (10umol/L) stimulation; the four hydrogenated pyrrolidine two thiocarbamate (PDTC) +IL-6 stimulation group, PDTC added to the culture medium, so that the final concentration is 10mmol/L, pretreatment of 1h after adding rhIL-6 (10ng/ml SB203580 (10umol/L) stimulation; 6) +IL-6 stimulation group, in the culture medium added SB203580 to a final concentration of 10umol/L, pretreatment of 1h after adding IL-6 (10ng/mL). All groups were incubated to stimulate the termination of the experiment, with 0,2,4,8,12,24h cells were collected.
3. the collected cells were extracted and the protein was extracted and the expression of EL was detected by Western blot.
More than 4. of the data were analyzed by statistics.
Result
1. Ang II can upregulate the expression of HUVECs EL. The expression of EL after.HUVECs stimulation at 4,8,12h is 0.362 + 0.041,0.738 + 0.034 and 0.387 + 0.051, which is significantly increased compared with 0h (0.254 + 0.027), all p0.05..
2. PDTC can inhibit the expression of EL induced by Ang II increased.HUVECs pretreatment with PDTC 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.287 + 0.037,0.433 + 0.041,0.303 + 0.025, and the expression of Ang II stimulation group in the corresponding period of EL was lower than that of p0.05.
3.SB203580 can inhibit the expression of EL induced by Ang II increased.HUVECs pretreatment with SB203580 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.258 + 0.027,0.372 + 0.038,0.296 + 0.034, and the expression of Ang II stimulation group in the corresponding period of EL was lower than that of p0.05.
4.IL-6 could upregulate the expression of HUVECs EL. The expression level of EL in.HUVECs stimulated by IL-6 was 0.293 + 0.062,0.633 + (0.052,0.462 + 0.065) at 4,8,12h, which was significantly increased compared with Oh (0.254 + 0.027).
5. PDTC can be inhibited by IL-6 induced expression of EL.HUVECs increased by pretreatment with PDTC 1h after adding Ang II stimulation, the EL expression in 4,8,12h was 0.208 + 0.056,0.582 + 0.036,0.428 + 0.066, and IL-6 stimulation group in the corresponding period of time compared to the amount of EL expression decreased obviously, p0.05.
6.SB203580 can be inhibited by IL-6 induced expression of EL.HUVECs increased by pretreatment with SB203580 1h after adding Ang II stimulation, the EL expression in 4,8,12,24h was 0.238 + 0.023,0.508 + 0.058,0.423 + 0.052,0.199 + 0.042, and IL-6 stimulation group in the corresponding period of EL expression was obviously lower than p0.05.
conclusion
1. Ang II may promote the expression of endothelial lipase in human umbilical vein endothelial cells through NF- kappa B and p38MAPK signal transduction pathway.
2.IL-6 may promote the expression of endothelial lipase in human umbilical vein endothelial cells through NF- kappa B and p38MAPK signal transduction pathway.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R363.2

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